Extended Data Fig. 4: Total SMAD2.3 quantification methodology.
From: Distinct pathways drive anterior hypoblast specification in the implanting human embryo
a, Examples from a segregated blastocyst presented in Fig. 2a of Epiblast (EPI), Hypoblast (HYPO) and Trophectoderm (TE) cells with nuclear (solid line) and cytoplasmic (dotted line) regions of interest (ROI) outlined. The measured nuclear (Avg. Nuc) and cytoplasmic (Avg. Cyto) and calculated nuclear/cytoplasmic ratio (N/C) of total SMAD2.3 intensity for individual cells is shown. b-c, Examples from a segregating blastocyst (b) and day 7 embryo (c) of cells with nuclear and cytoplasmic ROIs outlined. Measured nuclear, cytoplasmic, and calculated nuclear-to-cytoplasmic ratios of total SMAD2.3 intensities are shown for each cell. Note that here the raw, unprocessed SMAD2.3 signal is shown for the central 3-plane z-stacks used directly for quantification. N = 14 experiments. d-e, Examples of segregating (d) and segregated (e) human blastocysts. Inner cell mass cells co-expressing epiblast and hypoblast markers are marked with grey asterisks. N = 3 experiments. f-h, Violin plots depicting expression of NODAL receptors ACVR1B, ACVR2A, and ACVR2B across stages and lineages in human single cell RNA sequencing data. N = 9862 single cells. Scale bars: 100 µm.
